Project Summary/Abstract Past translational disappointments with stroke have tainted the perception of feasibility also for global cerebral ischemia (GCI). However, stroke and GCI are clearly distinct conditions. The translational potential in GCI is arguably much higher; yet, compared to stroke, much fewer efforts have been made to date (likely due to the smaller market share). GCI is caused by near- drowning or ?suffocation, as well as during heart bypass surgeries. However, most cases result from cardiac arrest (CA) followed by cardio-vascular resuscitation (CPR). Each year, approximately 600,000 Americans suffer from CA and receive CPR. Approximately 60% of the survivors have long-lasting moderate to severe cognitive impairments. Compared to stroke patients, CA/CPR patients are much more homogenous (duration of ischemia; time elapsed between ischemia and hospitalization; always full reperfusion at a well-defined time point). This is expected to provide a significant advantage in future clinical trials (as decreased variability significantly increases statistical power). Our preliminary studies have identified inhibitors that showed high therapeutic potential in a mouse model of CA/CPR that closely mimics the human condition. The inhibitors where injected i.v. at a clinically relevant timepoint after CA/CPR, and showed dramatic improvement. Such improvement was seen also over therapeutic hypothermia alone (the current standard of care); when both treatments were combined, only residual and barely detectable neuronal cell death was seen. In patients, cell death after 2-3 days (measured by serum levels of neuron- specific enolase) is strongly correlated with severity of the long-term outcome. Endpoints tested here additionally included functional effects on synaptic plasticity in the surviving neurons and behavioral outcome in a learning/memory task. Target validation has been done in mutant mice. Mode of target engagement has been characterized biochemically and structurally. The optimized lead compound is highly potent tight binder, with an in vitro IC50<0.4 nM and full therapeutic efficacy in vivo even at 0.01 mg/kg i.v.. The compound is highly selective, shows no hERG channel binding at the therapeutic concentration, and no apparent adverse health effects in mice even at 100fold of the therapeutic dose. Target-specific potential risk-factors have been evaluated in mice, and did not pose problems. Shelf-stability of the compound is very good. This project will collect the remaining data that are required for moving forward to IND- enabling safety studies, in the following four specific aims. Aim 1: Lead optimization through initial safety studies. We will initiate safety studies with a focus on the highest risk factors. If needed, this will be followed by optimization of the lead compound and/or the delivery route. Aim 2: Efficacy in a non-rodent model, specifically determined in a pig CA/CPR model that is established by local collaborators. Two initial doses will be used and efficacy will be compared to mouse based on PK comparison. Additional regimens will be determined based on the results of this and of the subsequent aims. Aim 3: PK and PK/PD relationship, will be determined in both test species after a single bolus and correlated to efficacy. Based on the outcome, booster treatment will be designed and tested. Functional target engagement will be verified by biochemical activity assays. Additionally, we will compare plasma protein binding between the test species and human. Aim 4: Optimization of the treatment regimen in mouse, by testing additional time points and if subsequent booster injections can further improve efficacy. Additionally, long-term functional outcomes after these treatments will be tested. The nature of the optimal treatment regimen will inform the required safety studies during the next development phase.